U.S. patent number 7,888,469 [Application Number 11/624,146] was granted by the patent office on 2011-02-15 for post-translation modification and clostridial neurotoxins.
This patent grant is currently assigned to Allergan, Inc.. Invention is credited to K. Roger Aoki, Ester Fernandez-Salas, Wei-Jen Lin, Athena Spanoyannis, Lance E. Steward.
United States Patent |
7,888,469 |
Steward , et al. |
February 15, 2011 |
**Please see images for:
( Certificate of Correction ) ** |
Post-translation modification and clostridial neurotoxins
Abstract
The present invention discloses modified neurotoxins with
altered biological persistence. In one embodiment, the modified
neurotoxins are derived from Clostridial botulinum toxins. Such
modified neurotoxins may be employed in treating various
conditions, including but not limited to muscular disorders,
hyperhidrosis, and pain.
Inventors: |
Steward; Lance E. (Irvine,
CA), Fernandez-Salas; Ester (Fullerton, CA), Spanoyannis;
Athena (Ashburn, VA), Aoki; K. Roger (Coto de Caza,
CA), Lin; Wei-Jen (Cerritos, CA) |
Assignee: |
Allergan, Inc. (Irvine,
CA)
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Family
ID: |
43598489 |
Appl.
No.: |
11/624,146 |
Filed: |
January 17, 2007 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100273986 A1 |
Oct 28, 2010 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11141513 |
May 31, 2005 |
7223577 |
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10004230 |
Oct 31, 2001 |
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Current U.S.
Class: |
530/350;
424/185.1 |
Current CPC
Class: |
C12Y
304/24069 (20130101); C12N 9/52 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
A61K
39/00 (20060101); C07K 1/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO96/39166 |
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Dec 1996 |
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WO |
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WO97/32599 |
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Sep 1997 |
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WO |
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WO98/07864 |
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Feb 1998 |
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WO |
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WO02/08268 |
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Jan 2002 |
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WO |
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Other References
Encinar, J. A. et al., Structural Stabilization of Botulinum
Neurotoxins by Tyrosine Phosphorylation, FEBS Lett. 429(1): 78-82
(1998). cited by other .
Ferrer-Montiel, A. V. et al., Tyrosine Phosphorylation Modulates
the Activity of Clostridial Neurotoxins, J. Biol. Chem. 271(31):
18322-18325 (1996). cited by other .
Keller, J. E. et al., Persistence of Botulinum Neurotoxin Action in
Cultured Spinal Cord Cells, FEBS Lett. 30;456(1): 137-142 (1999).
cited by other .
Kurazono, H. et al., Minimal Essential Domains Specifying Toxicity
of the Light Chains of Tetanus Toxin and Botulinum Neurotoxin Type
A, J. Biol. Chem. 267(21): 14751-14729 (1992). cited by other .
Raciborska D. A. and Charlton, M. P., Retention of Cleaved
Synaptosome-Associated Protein of 25 Kda (SNAP-25) in Neuromuscular
Junctions: A New Hypothesis to Explain Persistence of Botulinum A
Poisoning, Can. J. Physiol. Pharmacol. 77(9): 679-688 (1999). cited
by other .
Varshavsky, A., The N-End Rule: Funtions, Mysteries, Uses, Proc
Natl Acad Sci U S A. 93(22): 12142-12149 (1996). cited by other
.
Aubert et al, "Circular Dichrosim Studies of Synthetic
Asn-X-Ser/Thr-Containing Peptides: Structure-Glycosylation
Relationship", Archives of Biochemistry and Biophysics, vol. 208,
No. 1, pp. 20-29, 1981. cited by other .
Gavel et al, "Sequence differences between glycosylated and
non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for
protein engineering", Protein Engineering, vol. 3, No. 5, pp.
433-442, 1990. cited by other.
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Primary Examiner: Navarro; Mark
Attorney, Agent or Firm: Abel; Kenton Stathakis; Dean
Condino; Debra
Parent Case Text
This application is a continuation and claims priority pursuant to
35 U.S.C. .sctn.120 to U.S. patent application Ser. No. 11/141,513,
filed May 31, 2005, now U.S. Pat. No. 7,223,577, a continuation in
part application that claims priority pursuant to 35 U.S.C.
.sctn.120 to U.S. patent application Ser. No. 10/004,230, filed
Oct. 31, 2001 now abandoned, each which is hereby incorporated by
reference in its entirety.
Claims
What is claimed is:
1. A botulinum neurotoxin comprising a structural modification,
wherein the structural modification comprises at least one
additional N-glycosylation site, wherein the botulinum neurotoxin
can interfere with the functions of a neuron, and wherein the
additional N-glycosylation site increases biological persistence of
the botulinum neurotoxin relative to a naturally-occurring
botulinum neurotoxin of the same serotype without the additional
N-glycosylation site.
2. The botulinum toxin of claim 1, wherein the additional
N-glycosylation site is selected from the group consisting of SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26 and any combination thereof.
3. The botulinum neurotoxin of claim 1, wherein the botulinum
neurotoxin is selected from the group consisting of a botulinum
toxin type A, a botulinum toxin type B, a botulinum toxin type C1,
a botulinum toxin type D, a botulinum toxin type E, a botulinum
toxin type F and a botulinum toxin type G.
4. The botulinum neurotoxin of claim 3, wherein the botulinum
neurotoxin is a botulinum toxin type A.
5. The botulinum neurotoxin of claim 3, wherein the botulinum
neurotoxin is a botulinum toxin type C1.
6. The botulinum neurotoxin of claim 3, wherein the botulinum
neurotoxin is a botulinum toxin type E.
7. A botulinum neurotoxin comprising a proteolytic domain from the
light chain of a clostridial neurotoxin and at least one additional
N-glycosylation site, wherein the additional N-glycosylation site
increases biological persistence of the modified botulinum
neurotoxin relative to a naturally-occurring botulinum neurotoxin
of the same serotype without the additional N-glycosylation
site.
8. The botulinum toxin of claim 7, wherein the additional
N-glycosylation site is selected from the group consisting of SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 21,
SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID
NO: 26 and any combination thereof.
9. The botulinum neurotoxin of claim 7, wherein the botulinum
neurotoxin further comprises a H.sub.N fragment from the heavy
chain of a Clostridial neurotoxin.
10. The botulinum neurotoxin of claim 7, wherein the botulinum
neurotoxin further comprises a H.sub.C fragment derived from the
heavy chain of a Clostridial neurotoxin.
11. The botulinum neurotoxin of claim 7, wherein the botulinum
neurotoxin further comprises a H.sub.N fragment from the heavy
chain of a Clostridial neurotoxin and a H.sub.C fragment derived
from the heavy chain of a Clostridial neurotoxin.
Description
BACKGROUND
The present invention relates to modified neurotoxins, particularly
modified Clostridial neurotoxins, and use thereof to treat various
disorders, including neuromuscular disorders, autonomic nervous
system disorders and pain.
The clinical use of botulinum toxin serotype A (herein after
"BoNT/A"), a serotype of Clostridial neurotoxin, represents one of
the most dramatic role reversals in modern medicine: a potent
biologic toxin transformed into a therapeutic agent. BoNT/A has
become a versatile tool in the treatment of a wide variety of
disorders and conditions characterized by muscle hyperactivity,
autonomic nervous system hyperactivity and/or pain.
One of the reasons that BoNT/A has been selected over the other
serotypes, for example serotypes B, C.sub.1, D, E, F and G, for
clinical use is that BoNT/A has a substantially longer lasting
therapeutic effect. In other words, the inhibitory effect of BoNT/A
is more persistent. Therefore, the other serotypes of botulinum
toxins could potentially be effectively used in a clinical
environment if their biological persistence could be enhanced. For
example, parotoid sialocele is a condition where the patient
suffers from excessive salivation. Sanders et al. disclose in their
patent that serotype D may be very effective in reducing excessive
salivation. However, the biological persistence of serotype D
botulinum toxin is relatively short and thus may not be practical
for clinical use. If the biological persistence of serotype D may
be enhanced, it may effectively be used in a clinical environment
to treat, for example, parotid sialocele.
Another reason that BoNT/A has been a preferred neurotoxin for
clinical use is, as discussed above, its superb ability to
immobilize muscles through flaccid paralysis. For example, BoNT/A
is preferentially used to immobilize muscles and prevent limb
movements after a tendon surgery to facilitate recovery. However,
for some minor tendon surgeries, the healing time is relatively
short. It would be beneficial to have a BoNT/A without the
prolonged persistence for use in such circumstances so that the
patient can regain mobility at about the same time the recover from
the surgery.
Presently, the basis for the differences in persistence among the
various botulinum toxins is unknown. However, there are two main
theories explaining the differences in the persistence of the
toxins. Without wishing to be bound by any theory of operation or
mechanism of action, these theories will be discussed briefly
below. The first theory proposes that the persistence of a toxin
depends on which target protein and where on that target protein
that toxin attacks. Raciborska et al., Can. J. Physiol. Pharmcol.
77:679-688 (1999). For example, SNAP-25 and VAMP are proteins
required for vesicular docking, a necessary step for vesicular
exocytosis. BoNT/A cleaves the target protein SNAP-25 and BoNT/B
cleaves the target protein VAMP, respectively. The effect of each
is similar in that cleavage of either protein compromises the
ability of a neuron to release neurotransmitters via exocytosis.
However, damaged VAMP may be more easily replaced with new ones
that damaged SNAP-25, for example by replacement synthesis.
Therefore, since it takes longer for cells to synthesize new
SNAP-25 proteins to replace damaged ones, BoNT/A has longer
persistence. Id. At 685.
Additionally, the site of cleavage by a toxin may dictate how
quickly the damaged target proteins may be replaced. For example,
BoNT/A and E both cleave SNAP-25. However, they cleave at different
sites and BoNT/E causes shorter-lasting paralysis in patients,
compared with BoNT/A. Id. At 685-6.
The second theory proposes that the particular persistence of a
toxin depends on its particular intracellular half-life, or
stability, i.e., the longer the toxin is available in the cell, the
longer the effect. Keller et al., FEBS Letters 456:137-42 (1999).
Many factors contribute to the intracellular stability of a toxin,
but primarily, the better it is able to resist the metabolic
actions of intracellular proteases to break it down, the more
stable it is. Erdal et al. Naunyn-schmiedeber's Arch. Pharmacol.
351:67-78 (1995).
In general, the ability of a molecule to resist metabolic actions
of intracellular proteases may depend on its structures. For
example, the primary structure of a molecule may include a unique
primary sequence which may cause the molecule to be easily degraded
by proteases or difficult to be degraded. For example, Varshaysky
A. describes polypeptides terminating with certain amino acids are
more susceptible to degrading proteases. Proc. Natl. Acad. Sci. USA
93:12142-12149 (1996).
Furthermore, intracellular enzymes are known to modify molecules,
for example polypeptides through, for example, N-glycosylation,
phosphorylation etc. this kind of modification will be referred to
herein as "secondary modification". "Secondary modification" often
refers to the modification of endogenous molecules, for example,
polypeptides after they are translated from RNAs. However, as used
herein, "secondary modification" may also refer to an enzyme's, for
example an intracellular enzyme's, ability to modify exogenous
molecules. For example, after a patient is administered with
exogenous molecules, e.g. drugs, these molecules may undergo a
secondary modification by the action of the patient's enzymes, for
example intracellular enzymes.
Certain secondary modifications of molecules, for example
polypeptides, may resist or facilitate the actions of degrading
proteases. These secondary modifications may, among other things,
(1) affect the ability of a degrading protease to act directly on
the molecule and/or (2) affect the ability of the molecules to be
sequestered into vesicles to be protected against these degrading
proteases.
There is a need to have modified neurotoxins which have efficacies
of the various botulinum toxin serotypes, but with altered
biological persistence, and methods for preparing such toxins.
SUMMARY OF THE INVENTION
The present invention meets this need and provides for modified
neurotoxins with altered biological persistence and methods for
preparing such toxins.
Without wishing to be limited by any theory or mechanism of
operation, it is believed that Botulinum toxins have secondary
modification sites, which may determine their biological
persistence. A "secondary modification site" as used herein means a
location on a molecule, for example a particular fragment or a
polypeptide, which may be targeted by an enzyme, for example an
intra-cellular enzyme, to affect a modification to the site, for
example phosphorylation, glycosylation, etc. The secondary
modification, for example phosphorylation, may help resist or
facilitate the actions of degrading proteases acting on the toxins,
which in turn increase or decrease the persistence, or stability,
of the toxins, respectively. Alternatively, it is believed that
these secondary modification sites may prevent or facilitate the
transportation of the toxin into vesicles to be protected from
degrading proteases. It is further believed that one of the roles
of the secondary modification is to add to or take away the three
dimensional and/or the chemical requirements necessary for protein
interactions, for example between a molecule and a degrading
protease, or a molecule and a vesicular transporter.
Therefore, a modified neurotoxin including a structural
modification may have altered persistence as compared to an
identical neurotoxin without the structural modification. The
structural modification may include a partial or complete deletion
or mutation of at least one modification site. Alternatively, the
structural modification may include the addition of a certain
modification site. In one embodiment, the altered persistence is
the enhancement of the biological persistence. In another
embodiment, the altered persistence is the reduction of biological
persistence. Preferably, the altered persistence is affected by the
alteration in the stability of the modified neurotoxin.
For example, the light chain of BoNT/A has amino acid fragments for
various secondary modification sites (hereinafter "modification
sites") including, but not limited to, N-glycosylation, casein
kinase II (CK-2) phosphorylation, N-terminal myristylation, protein
kinase C (PKC) phosphorylation and tyrosine phosphorylation. BoNT/E
also has these various secondary modification sites. The structural
modification includes the deletion or mutation of one or more of
these secondary modification sites. The structural modification may
also include the addition of one or more of a modification site to
a neurotoxin to form a modified neurotoxin.
This invention also provide for methods of producing modified
neurotoxins. Additionally, this invention provide for methods of
using the modified neurotoxins to treat biological disorders.
Definitions
Before proceeding to describe the present invention, the following
definitions are provided and apply herein.
"Heavy chain" means the heavy chain of a clostridial neurotoxin. It
preferably has a molecular weight of about 100 kD and may be
referred to herein as H chain or as H.
"H.sub.N" means a fragment (preferably having a molecular weight of
about 50 kD) derived from the H chain of a Clostridial neurotoxin
which is approximately equivalent to the amino terminal segment of
the H chain, or the portion corresponding to that fragment in the
intact in the H chain. It is believed to contain the portion of the
natural or wild type clostridial neurotoxin involved in the
translocation of the L chain across an intracellular endosomal
membrane.
"H.sub.C" means a fragment (about 50 kD) derived from the H chain
of a clostridial neurotoxin which is approximately equivalent to
the carboxyl terminal segment of the H chain, or the portion
corresponding to that fragment in the intact H chain. It is
believed to be immunogenic and to contain the portion of the
natural or wild type Clostridial neurotoxin involved in high
affinity, presynaptic binding to motor neurons.
"Light chain" means the light chain of a clostridial neurotoxin. It
preferably has a molecular weight of about 50 kD, and can be
referred to as L chain, L or as the proteolytic domain (amino acid
sequence) of a clostridial neurotoxin. The light chain is believed
to be effective as an inhibitor of neurotransmitter release when it
is released into a cytoplasm of a target cell.
"Neurotoxin" means a molecule that is capable of interfering with
the functions of a neuron. The "neurotoxin" may be naturally
occurring or man-made.
"Modified neurotoxin" means a neurotoxin which includes a
structural modification. In other words, a "modified neurotoxin" is
a neurotoxin which has been modified by a structural modification.
The structural modification changes the biological persistence,
preferably the biological half-life, of the modified neurotoxin
relative to the neurotoxin from which the modified neurotoxin is
made. The modified neurotoxin is structurally different from a
naturally existing neurotoxin.
"Structural modification" means a physical change to the neurotoxin
that may be affected by, for example, covalently fusing one or more
amino acids to the neurotoxin. "Structural modification" also means
the deletion of one or more amino acids from a neurotoxin.
Furthermore, "structural modification" may also mean any changes to
a neurotoxin that makes it physically or chemically different from
an identical neurotoxin without the structural modification.
"Biological persistence" means the time duration in which a
neurotoxin or a modified neurotoxin causes an interference with a
neuronal function, for example the time duration in which a
neurotoxin or a modified neurotoxin causes a substantial inhibition
of the release of acetylcholine from a nerve terminal.
"Biological half-life" means the time that the concentration of a
neurotoxin or a modified neurotoxin, preferably the active portion
of the neurotoxin or modified neurotoxin, for example the light
chain of botulinum toxins, is reduced to half of the original
concentration in a mammal, preferably in the neurons of the
mammal.
"Modification site" means a particular amino acid or a fragment of
amino acids where upon secondary modification may takes place.
"Modification site" may also mean a particular amino acid or a
particular fragment of amino acids necessary for a certain
secondary modification to occur.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is, in part, based upon the discovery that
the biological persistence of a neurotoxin may be altered by
structurally modifying the neurotoxin. In other words, a modified
neurotoxin with an altered biological persistence may be formed
from a neurotoxin containing or including a structural
modification. Preferably, the inclusion of the structural
modification may alter the biological half-life of the modified
neurotoxin. An altered biological persistence, preferably an
altered biological half-life, means that the biological persistence
(or biological half-life) of a modified neurotoxin is different
from that of an identical neurotoxin without the structural
modification. Additionally, the biological persistence, preferably
the biological half-life, may be altered to be longer or
shorter.
In one embodiment, the structural modification includes a partial
or complete deletion or mutation of the modification site of the
neurotoxin to form a modified neurotoxin. The inclusion of the
modification site may enhance the biological persistence of the
modified neurotoxin. Preferably, the partial or complete deletion,
or mutation of the modification site enhances the biological
half-life of the modified neurotoxin. More preferably, the
biological half-life of the modified neurotoxin is enhanced by
about 10%. Even more preferably, the biological half-life of the
modified neurotoxin is enhanced by about 100%. Generally speaking,
the modified neurotoxin has a biological persistence of about 20%
to 300% more than an identical neurotoxin without the structural
modification. That is, for example, the modified neurotoxin
including the modified modification site is able to cause a
substantial inhibition of acetylcholine release from a nerve
terminal for about 20% to about 300% longer than a neurotoxin that
is not modified.
In one embodiment, the structural modification includes a partial
or complete deletion or mutation of the modification site of the
neurotoxin to form a modified neurotoxin. The inclusion of the
modification site may reduce the biological persistence of the
modified neurotoxin. Preferably, the partial or complete deletion,
or mutation of the modification site reduces the biological
half-life of the modified neurotoxin. More preferably, the
biological half-life of the modified neurotoxin is reduced by about
10%. Even more preferably, the biological half-life of the modified
neurotoxin is reduced by about 99%. Generally speaking, the
modified neurotoxin has a biological persistence of about 20% to
300% less than an identical neurotoxin without the structural
modification. That is, for example, the modified neurotoxin
including the modified modification site is able to cause a
substantial inhibition of acetylcholine release from a nerve
terminal for about 20% to about 300% shorter in time than a
neurotoxin that is not modified.
For example, BoNT/A and BoNT/E have the following potential
secondary modification sites as shown on Tables 1 and 2,
respectively.
TABLE-US-00001 TABLE 1 N-glycosylation sites 173-NLTR (SEQ ID NO:
1) 382-NYTI (SEQ ID NO: 2) 411-NFTK (SEQ ID NO: 3) 417-NFTG (SEQ ID
NO: 4) Casein kinase II (CK-2) phosphorylation sites 51-TNPE (SEQ
ID NO: 5) 70-SYYD (SEQ ID NO: 6) 79-TDNE (SEQ ID NO: 7) 120-STID
(SEQ ID NO: 8) 253-SGLE (SEQ ID NO: 9) 258-SFEE (SEQ ID NO: 10)
275-SLQE (SEQ ID NO: 11) 384-TIYD (SEQ ID NO: 12) N-terminal
myristylation sites 15-GVDIAY (SEQ ID NO: 13) 141-GSYRSE (SEQ ID
NO: 14) 254-GLEVSF (SEQ ID NO: 15) Protein kinase C (PKC)
phosphorylation sites 142-SYR (SEQ ID NO: 16) 327-SGK (SEQ ID NO:
17) 435-TSK (SEQ ID NO: 18) Tyrosine phosphorylation sites
92-KLFERIY (SEQ ID NO: 19) 334-KLKFDKLY (SEQ ID NO: 20)
TABLE-US-00002 TABLE 2 N-glycosylation sites 97-NLSG (SEQ ID NO:
21) 138-NGSG (SEQ ID NO: 22) 161-NSSN (SEQ ID NO: 23) 164-NISL (SEQ
ID NO: 24) 365-NDSI (SEQ ID NO: 25) 370-NISE (SEQ ID NO: 26) Casein
kinase II (CK-2) phosphorylation sites. 51-TPQD (SEQ ID NO: 27)
67-SYYD (SEQ ID NO: 28) 76-SDEE (SEQ ID NO: 29) 130-SAVE (SEQ ID
NO: 30) 198-SMNE (SEQ ID NO: 31) 247-TNIE (SEQ ID NO: 32) 333-SFTE
(SEQ ID NO: 33) 335-TEFD (SEQ ID NO: 34) N-terminal myristylation
sites 220-GLYGAK (SEQ ID NO: 35) 257-GTDLNI (SEQ ID NO: 36)
386-GQNANL (SEQ ID NO: 37) Protein kinase C (PKC) phosphorylation
sites 60-SLK (SEQ ID NO: 38) 166-SLR (SEQ ID NO: 39) 191-SFR (SEQ
ID NO: 40) 228-TTK (SEQ ID NO: 41) 234-TQK (SEQ ID NO: 42) 400-TGR
(SEQ ID NO: 43) 417-SVK (SEQ ID NO: 44) Tyrosine kinase
phosphorylation sites 62-KNGDSSY (SEQ ID NO: 45) 300-KDVFEAKY (SEQ
ID NO: 46)
In one preferred embodiment, one or more of the modification site
of BoNT/A, for example the N-glycosylation site, is partially
deleted, completely deleted or mutated, resulting in a modified
neurotoxin with an altered biological persistence, preferably an
altered biological half-life. In one embodiment, the modified
neurotoxin is altered to have a longer biological persistence,
preferably longer biological half-life. In another embodiment, the
modified neurotoxin is altered to have a shorter persistence,
preferably a shorter biological half-life.
In one preferred embodiment, one or more of the modification site
of BoNT/E, for example the N-glycosylation site, is partially
deleted, completely deleted or mutated, resulting in a modified
neurotoxin with an altered biological persistence, preferably an
altered biological half-life. In one embodiment, the modified
neurotoxin is altered to have a longer biological persistence,
preferably longer biological half-life. In another embodiment, the
modified neurotoxin is altered to have a shorter persistence,
preferably a shorter biological half-life as compared to an
identical neurotoxin without the structural modification.
In one broad embodiment, the modified neurotoxin may include
additional modification sites fused onto neurotoxins to form
modified neurotoxins. The modification sites may be any
modification sites known in the art, including the ones listed on
Tables 1 and 2. In one embodiment, such inclusion of the
modification site may enhance the biological persistence of the
modified neurotoxin. Preferably, the modification site enhances the
biological half-life of the modified neurotoxin. More preferably,
the biological half-life of the modified neurotoxin is enhanced by
about 10%. Even more preferably, the biological half-life of the
modified neurotoxin is enhanced by about 100%. Generally speaking,
the modified neurotoxin has a biological persistence of about 20%
to 300% more than an identical neurotoxin without the structural
modification. That is, for example, the modified neurotoxin
including the modified site is able to cause a substantial
inhibition of acetylcholine release from a nerve terminal for about
20% to about 300% longer than a neurotoxin that is not modified. A
non-limiting example of a modified neurotoxin with an additional
modification site is Bo/E with a casein kinase II phosphorylation
site, preferably TDNE, fused to its primary structure. More
preferably, the TDNE is fused to position 79 of BoNT/E or a
position on BoNT/E which substantially corresponds to position 79
of BoNT/A.
In one broad embodiment, the modified neurotoxin may include
additional modification sites fused onto neurotoxins to form
modified neurotoxins. The modification sites may be any
modification sites known in the art, including the ones listed on
Tables 1 and 2. In one embodiment, such inclusion of the
modification site may reduce the biological persistence of the
modified neurotoxin. Preferably, the modification site reduces the
biological half-life of the modified neurotoxin. More preferably,
the biological half-life of the modified neurotoxin is reduced by
about 10%. Even more preferably, the biological half-life of the
modified neurotoxin is reduced by about 99%. Generally speaking,
the modified neurotoxin has a biological persistence of about 20%
to 300% less than an identical neurotoxin without the structural
modification. That is, for example, the modified neurotoxin
including the modified site is able to cause a substantial
inhibition of acetylcholine release from a nerve terminal for about
20% to about 300% shorter in time than a neurotoxin that is not
modified. A non-limiting example of a modified neurotoxin with an
additional modification site is Bo/A with a casein kinase II
phosphorylation site, preferably SDEE, fused to its primary
structure. More preferably, the SDEE is fused to position 76 of
BoNT/A or a position on BoNT/A which substantially corresponds to
position 76 of BoNT/E.
In one embodiment, the structural modification may include the
addition and the partial or complete deletion or mutation of
modification sites. For example, a modified neurotoxin may be
BoNT/A with GVDIAY at position 15 deleted and includes a SLK
fragment for protein kinase C phosphorylation. The SLK fragment is
preferably fused to position 60 of BoNT/A or a position on BoNT/A
which substantially corresponds to position 60 of BoNT/E. The
modified neurotoxin according to this embodiment may have altered
biological persistence. In one embodiment, the biological
persistence is increased. In another embodiment, the biological
persistence is decreased. Preferably, the modified neurotoxin
according to this embodiment may have altered biological half-life.
In one embodiment, the biological half-life is increased. In
another embodiment, the biological half-life is decreased.
In one broad aspect of the present invention, a method is provided
for treating a biological disorder using a modified neurotoxin. The
treatments may include treating neuromuscular disorders, autonomic
nervous system disorders and pain.
The neuromuscular disorders and conditions that may be treated with
a modified neurotoxin include: for example, strabismus,
blepharospasm, spasmodic torticollis (cervical dystonia),
oromandibular dystonia and spasmodic dysphonia (laryngeal
dystonia).
For example, Borodic U.S. Pat. No. 5,053,005 discloses methods for
treating juvenile spinal curvature, i.e. scoliosis, using BoNT/A.
The disclosure of Borodic is incorporated in its entirety herein by
reference. In one embodiment, using substantially similar methods
as disclosed by Borodic, a modified neurotoxin is administered to a
mammal, preferably a human, to treat spinal curvature. In a
preferred embodiment, a modified neurotoxin comprising BoNT/E fused
with an N-terminal myristylation site is administered. Even more
preferably, a modified neurotoxin comprising BoNT/E with an
N-terminal myristylation site fused to position 15 of its light
chain, or a position substantially corresponding to position 15 of
the BoNT/A light chain, is administered to the mammal, preferably a
human, to treat spinal curvature. The modified neurotoxin may be
administered to treat other neuromuscular disorders using well
known techniques that are commonly performed with BoNT/A.
Autonomic nervous system disorders may also be treated with a
modified neurotoxin. For example, glandular malfunctioning is an
autonomic nervous system disorder. Glandular malfunctioning
includes excessive sweating and excessive salivation. Respiratory
malfunctioning is another example of an autonomic nervous system
disorder. Respiratory malfunctioning includes chronic obstructive
pulmonary disease and asthma. Sanders et al. discloses methods for
treating the autonomic nervous system, such as excessive sweating,
excessive salivation, asthma, etc., using naturally existing
botulinum toxins. The disclosure of Sander et al. is incorporated
in its entirety by reference herein. In one embodiment,
substantially similar methods to that of Sanders et al. may be
employed, but using a modified neurotoxin, to treat autonomic
nervous system disorders such as the ones discussed above. For
example, a modified neurotoxin may be locally applied to the nasal
cavity of the mammal in an amount sufficient to degenerate
cholinergic neurons of the autonomic nervous system that control
the mucous secretion in the nasal cavity.
Pain that may be treated by a modified neurotoxin includes pain
caused by muscle tension, or spasm, or pain that is not associated
with muscle spasm. For example, Binder in U.S. Pat. No. 5,714,468
discloses that headache caused by vascular disturbances, muscular
tension, neuralgia and neuropathy may be treated with a naturally
occurring botulinum toxin, for example BoNT/A. The disclosure of
Binder is incorporated in its entirety herein by reference. In one
embodiment, substantially similar methods to that of Binder may be
employed, but using a modified neurotoxin, to treat headache,
especially the ones caused by vascular disturbances, muscular
tension, neuralgia and neuropathy. Pain caused by muscle spasm may
also be treated by an administration of a modified neurotoxin. For
example, a modified neurotoxin comprising BoNT/E with an N-terminal
myristylation site fused to position 15 of its light chain, or a
position substantially corresponding to position 15 of the BoNT/A
light chain, may be administered intramuscularly at the pain/spasm
location to alleviate pain.
Furthermore, a modified neurotoxin may be administered to a mammal
to treat pain that is not associated with a muscular disorder, such
as spasm. In one broad embodiment, methods of the present invention
to treat non-spasm related pain include central administration or
peripheral administration of the modified neurotoxin.
For example, Foster et al. in U.S. Pat. No. 5,989,545 discloses
that a botulinum toxin conjugated with a targeting moiety may be
administered centrally (intrathecally) to alleviate pain. The
disclosure of Foster et al. is incorporated in its entirety by
reference herein. In one embodiment, substantially similar methods
to that of Foster et al. may be employed, but using the modified
neurotoxin according to this invention, to treat pain. The pain to
be treated may be an acute pain, or preferably, chronic pain.
An acute or chronic pain that is not associated with a muscle spasm
may also be alleviated with a local, peripheral administration of
the modified neurotoxin to an actual or a perceived pain location
on the mammal. In one embodiment, the modified neurotoxin is
administered subcutaneously at or near the location of pain, for
example at or near a cut. In another embodiment, the modified
neurotoxin is administered intramuscularly at or near the location
of pain, for example at or near a bruise location on the mammal. In
another embodiment, the modified neurotoxin is injected directly
into a joint of a mammal, for treating or alleviating pain cause
arthritis conditions. Also, frequent repeated injections or
infusion of the modified neurotoxin to a peripheral pain location
is within the scope of the present invention. However, given the
long lasting therapeutic effects of the present invention, frequent
injections or infusion of the neurotoxin may not be necessary. For
example, practice of the present invention can provide an analgesic
effect, per injection, for 2 months or longer, for example 27
months, in humans.
Without wishing to limit the invention to any mechanism or theory
of operation, it is believed that when the modified neurotoxin is
administered locally to a peripheral location, it inhibits the
release of neuro-substances, for example substance P, from the
peripheral primary sensory terminal. Since the release of substance
P by the peripheral primary sensory terminal may cause or at least
amplify pain transmission process, inhibition of its release at the
peripheral primary sensory terminal will dampen the transmission of
pain signals from reaching the brain.
In addition to having pharmacologic actions at the peripheral
location, the modified neurotoxin of the present invention may also
have inhibitory effects in the central nervous system. Presumably
the retrograde transport is via the primary afferent. This
hypothesis is supported by our experimental data which shows that
BoNT/A is retrograde transported to the dorsal horn when the
neurotoxin is injected peripherally. Moreover, work by Weigand et
al, Nauny-Schmiedeberg's Arch. Pharmacol. 1976; 292, 161-165, and
Habermann, Nauny-Schmiedeberg's Arch. Pharmacol. 1974; 281, 47-56,
showed that botulinum toxin is able to ascend to the spinal area by
retrograde transport. As such, a modified neurotoxin, for example
BoNT/A with one or more amino acids deleted from the leucine-based
motif, injected at a peripheral location, for example
intramuscularly, may be retrograde transported from the peripheral
primary sensory terminal to the central primary sensory
terminal.
The amount of the modified neurotoxin administered can vary widely
according to the particular disorder being treated, its severity
and other various patient variables including size, weight, age,
and responsiveness to therapy. Generally, the dose of modified
neurotoxin to be administered will vary with the age, presenting
condition and weight of the mammal, preferably a human, to be
treated. The potency of the modified neurotoxin will also be
considered.
Assuming a potency which is substantially equivalent to
LD.sub.50=2,730 U in a human patient and an average person is 75
kg, a lethal dose would be about 36 U/kg of a modified neurotoxin.
Therefore, when a modified neurotoxin with such an LD.sub.50 is
administered, it would be appropriate to administer less than 36
U/kg of the modified neurotoxin into human subjects. Preferably,
about 0.01 U/kg to 30 U/kg of the modified neurotoxin is
administered. More preferably, about 1 U/kg to about 15 U/kg of the
modified neurotoxin is administered. Even more preferably, about 5
U/kg to about 10 U/kg modified neurotoxin is administered.
Generally, the modified neurotoxin will be administered as a
composition at a dosage that is proportionally equivalent to about
2.5 cc/100 U. Those of ordinary skill in the art will know, or can
readily ascertain, how to adjust these dosages for neurotoxin of
greater or lesser potency.
Although examples of routes of administration and dosages are
provided, the appropriate route of administration and dosage are
generally determined on a case by case basis by the attending
physician. Such determinations are routine to one of ordinary skill
in the art (see for example, Harrison's Principles of Internal
Medicine (1998), edited by Anthony Fauci et al., 14.sup.th edition,
published by McGraw Hill). For example, the route and dosage for
administration of a modified neurotoxin according to the present
disclosed invention can be selected based upon criteria such as the
solubility characteristics of the modified neurotoxin chosen as
well as the types of disorder being treated.
The modified neurotoxin may be produced by chemically linking the
modification sites to a neurotoxin using conventional chemical
methods well known in the art. The neurotoxin may be obtained from
harvesting neurotoxins. For example, BoNT/E can be obtained by
establishing and growing cultures of Clostridium botulinum in a
fermenter and then harvesting and purifying the fermented mixture
in accordance with known procedures. All the botulinum toxin
serotypes are initially synthesized as inactive single chain
proteins which must be cleaved or nicked by proteases to become
neuroactive. The bacterial strains that make botulinum toxin
serotypes A and G possess endogenous proteases and serotypes A and
G can therefore be recovered from bacterial cultures in
predominantly their active form. In contrast, botulinum toxin
serotypes C.sub.1, D and E are synthesized by nonproteolytic
strains and are therefore typically unactivated when recovered from
culture. Serotypes B and F are produced by both proteolytic and
nonproteolytic strains and therefore can be recovered in either the
active or inactive form. However, even the proteolytic strains that
produce, for example, the BoNT/B serotype only cleave a portion of
the toxin produced. The exact proportion of nicked to unnicked
molecules depends on the length of incubation and the temperature
of the culture. Therefore, a certain percentage of any preparation
of, for example, the BoNT/B toxin is likely to be inactive,
possibly accounting for the known significantly lower potency of
BoNT/B as compared to BoNT/A. The presence of inactive botulinum
toxin molecules in a clinical preparation will contribute to the
overall protein load of the preparation, which has been linked to
increased antigenicity, without contributing to its clinical
efficacy. Additionally, it is known that BoNT/B has, upon
intramuscular injection, a shorter duration of activity and is also
less potent than BoNT/A at the same dose level.
The modified neurotoxin may also be produced by recombinant
techniques. Recombinant techniques are preferable for producing a
neurotoxin having amino acid sequence regions from different
Clostridial species or having modified amino acid sequence regions.
Also, the recombinant technique is preferable in producing BoNT/A
with the modified (deleted or mutated) or added modification sites.
The technique includes steps of obtaining genetic materials from
natural sources, or synthetic sources, which have codes for a
neuronal binding moiety, an amino acid sequence effective to
translocate the neurotoxin or a part thereof, and an amino acid
sequence having therapeutic activity when released into a cytoplasm
of a target cell, preferably a neuron. In a preferred embodiment,
the genetic materials have codes for the biological persistence
enhancing component, preferably the leucine-based motif, the
H.sub.C, the H.sub.N and the L chain of the Clostridial neurotoxins
and fragments thereof. The genetic constructs are incorporated into
host cells for amplification by first fusing the genetic constructs
with a cloning vectors, such as phages or plasmids. Then the
cloning vectors are inserted into hosts, preferably E. coli's.
Following the expressions of the recombinant genes in host cells,
the resultant proteins can be isolated using conventional
techniques.
There are many advantages to producing these modified neurotoxins
recombinantly. For example, to form a modified neurotoxin, a
modifying fragment must be attached or inserted into a neurotoxin.
The production of neurotoxin from anaerobic Clostridium cultures is
a cumbersome and time-consuming process including a multi-step
purification protocol involving several protein precipitation steps
and either prolonged and repeated crystallization of the toxin or
several stages of column chromatography. Significantly, the high
toxicity of the product dictates that the procedure must be
performed under strict containment (BL-3). During the fermentation
process, the folded single-chain neurotoxins are activated by
endogenous clostridial proteases through a process termed nicking
to create a dichain. Sometimes, the process of nicking involves the
removal of approximately 10 amino acid residues from the
single-chain to create the dichain form in which the two chains
remain covalently linked through the intrachain disulfide bond.
The nicked neurotoxin is much more active than the unnicked form.
The amount and precise location of nicking varies with the
serotypes of the bacteria producing the toxin. The differences in
single-chain neurotoxin activation and, hence, the yield of nicked
toxin, are due to variations in the serotype and amounts of
proteolytic activity produced by a given strain. For example,
greater than 99% of Clostridial botulinum serotype A single-chain
neurotoxin is activated by the Hall A Clostridial botulinum strain,
whereas serotype B and E strains produce toxins with lower amounts
of activation (0 to 75% depending upon the fermentation time).
Thus, the high toxicity of the mature neurotoxin plays a major part
in the commercial manufacture of neurotoxins as therapeutic
agents.
The degree of activation of engineered clostridial toxins is,
therefore, an important consideration for manufacture of these
materials. It would be a major advantage if neurotoxins such as
botulinum toxin and tetanus toxin could be expressed,
recombinantly, in high yield in rapidly-growing bacteria (such as
heterologous E. coli cells) as relatively non-toxic single-chains
(or single chains having reduced toxic activity) which are safe,
easy to isolate and simple to convert to the fully-active form.
With safety being a prime concern, previous work has concentrated
on the expression in E. coli and purification of individual H and L
chains of tetanus and botulinum toxins; these isolated chains are,
by themselves, non-toxic; see Li et al., Biochemistry 33:7014-7020
(1994); Zhou et al., Biochemistry 34:15175-15181 (1995), hereby
incorporated by reference herein. Following the separate production
of these peptide chains and under strictly controlled conditions
the H and L chains can be combined by oxidative disulphide linkage
to form the neuroparalytic di-chains(di-polypeptide), linked
together by a disulfide bond. Preferably one of the polypeptides is
a Clostridial neurotoxin heavy chain and the other is a Clostridial
neurotoxin light chain. The neuronal binding moiety is preferably
part of the heavy chain.
EXAMPLES
The following non-limiting examples provide those of ordinary skill
in the art with specific preferred methods to treat non-spasm
related pain within the scope of the present invention and are not
intended to limit the scope of the invention.
Example 1
Treatment of Pain Associated with Muscle Disorder
An unfortunate 36 year old woman has a 15 year history of
temporomandibular joint disease and chronic pain along the masseter
and temporalis muscles. Fifteen years prior to evaluation she noted
increased immobility of the jaw associated with pain and jaw
opening and closing and tenderness along each side of her face. The
left side is originally thought to be worse than the right. She is
diagnosed as having temporomandibular joint (TMJ) dysfunction with
subluxation of the joint and is treated with surgical orthoplasty
meniscusectomy and condyle resection.
She continues to have difficulty with opening and closing her jaw
after the surgical procedures and for this reason, several years
later, a surgical procedure to replace prosthetic joints on both
sides is performed. After the surgical procedure progressive spasms
and deviation of the jaw ensues. Further surgical revision is
performed subsequent to the original operation to correct
prosthetic joint loosening. The jaw continues to exhibit
considerable pain and immobility after these surgical procedures.
The TMJ remained tender as well as the muscle itself. There are
tender points over the temporomandibular joint as well as increased
tone in the entire muscle. She is diagnosed as having post-surgical
myofascial pain syndrome and is injected with about 8 U/kg to about
15 U/kg of the modified neurotoxin into the masseter and temporalis
muscles, preferably the modified neurotoxin comprises BoNT/E with
an N-terminal myristylation site, for example GVDIAY, fused to
position 15 of its light chain, or a position substantially
corresponding to position 15 of the BoNT/A light chain.
Several days after the injections she noted substantial improvement
in her pain and reports that her jaw feels looser. This gradually
improves over a 2 to 3 week period in which she notes increased
ability to open the jaw and diminishing pain. The patient states
that the pain is better than at any time in the last 4 years. The
improved condition persists for up to 27 months after the original
injection of the modified neurotoxin.
Example 2
Treatment of Pain Subsequent to Spinal Cord Injury
A patient, age 39, experiencing pain subsequent to spinal cord
injury is treated by intrathecal administration, for example by
spinal tap or by catherization (for infusion), to the spinal cord,
with about 0.1 U/kg to about 10 U/kg of the modified neurotoxin,
preferably the modified neurotoxin comprises BoNT/E with an
N-terminal myristylation site, for example GVDIAY, fused to
position 15 of its light chain, or a position substantially
corresponding to position 15 of the BoNT/A light chain. The
particular toxin dose and site of injection, as well as the
frequency of toxin administrations depend upon a variety of factors
within the skill of the treating physician, as previously set
forth. Within about 1 to about 7 days after the modified neurotoxin
administration, the patient's pain is substantially reduced. The
pain alleviation persists for up to 27 months.
Example 3
Peripheral Administration of a Modified Neurotoxin to Treat
"Shoulder-Hand Syndrome"
Pain in the shoulder, arm, and hand can develop, with muscular
dystrophy, osteoporosis, and fixation of joints. While most common
after coronary insufficiency, this syndrome may occur with cervical
osteoarthritis or localized shoulder disease, or after any
prolonged illness that requires the patient to remain in bed.
A 46 year old woman presents a shoulder-hand syndrome type pain.
The pain is particularly localized at the deltoid region. The
patient is treated by a bolus injection of about 0.05 U/kg to about
2 U/kg of a modified neurotoxin subcutaneously to the shoulder,
preferably the modified neurotoxin comprises BoNT/E with an
N-terminal myristylation site, for example GVDIAY, fused to
position 15 of its light chain, or a position substantially
corresponding to position 15 of the BoNT/A light chain. The
particular dose as well as the frequency of administrations depends
upon a variety of factors within the skill of the treating
physician, as previously set forth. Within 1-7 days after modified
neurotoxin administration the patient's pain is substantially
alleviated. The duration of the pain alleviation is from about 7 to
about 27 months.
Example 4
Peripheral Administration of a Modified Neurotoxin to Treat
Postherpetic Neuralgia
Postherpetic neuralgia is one of the most intractable of chronic
pain problems. Patients suffering this excruciatingly painful
process often are elderly, have debilitating disease, and are not
suitable for major interventional procedures. The diagnosis is
readily made by the appearance of the healed lesions of herpes and
by the patient's history. The pain is intense and emotionally
distressing. Postherpetic neuralgia may occur anywhere, but is most
often in the thorax.
A 76 year old man presents a postherpetic type pain. The pain is
localized to the abdomen region. The patient is treated by a bolus
injection of between about 0.05 U/kg to about 2 U/kg of a modified
neurotoxin intradermally to the abdomen, preferably the modified
neurotoxin comprises BoNT/E with an N-terminal myristylation site,
for example GVDIAY, fused to position 15 of its light chain, or a
position substantially corresponding to position 15 of the BoNT/A
light chain. The particular dose as well as the frequency of
administrations depends upon a variety of factors within the skill
of the treating physician, as previously set forth. Within 1-7 days
after modified neurotoxin administration the patient's pain is
substantially alleviated. The duration of the pain alleviation is
from about 7 to about 27 months.
Example 5
Peripheral Administration of a Modified Neurotoxin to Treat
Nasopharyngeal Tumor Pain
These tumors, most often squamous cell carcinomas, are usually in
the fossa of Rosenmuller and may invade the base of the skull. Pain
in the face is common. It is constant, dull-aching in nature.
A 35 year old man presents a nasopharyngeal tumor type pain. Pain
is found at the lower left cheek. The patient is treated by a bolus
injection of between about 0.05 U/kg to about 2 U/kg of a modified
neurotoxin intramuscularly to the cheek, preferably the modified
neurotoxin comprises BoNT/E with an N-terminal myristylation site,
for example GVDIAY, fused to position 15 of its light chain, or a
position substantially corresponding to position 15 of the BoNT/A
light chain. The particular dose as well as the frequency of
administrations depends upon a variety of factors within the skill
of the treating physician, as previously set forth. Within 1-7 days
after modified neurotoxin administration the patient's pain is
substantially alleviated. The duration of the pain alleviation is
from about 7 to about 27 months.
Example 6
Peripheral Administration of a Modified Neurotoxin to Treat
Inflammatory Pain
A patient, age 45, presents an inflammatory pain in the chest
region. The patient is treated by a bolus injection of between
about 0.05 U/kg to about 2 U/kg of a modified neurotoxin
intramuscularly to the chest, preferably the modified neurotoxin
comprises BoNT/E with an N-terminal myristylation site, for example
GVDIAY, fused to position 15 of its light chain, or a position
substantially corresponding to position 15 of the BoNT/A light
chain. The particular dose as well as the frequency of
administrations depends upon a variety of factors within the skill
of the treating physician, as previously set forth. Within 1-7 days
after modified neurotoxin administration the patient's pain is
substantially alleviated. The duration of the pain alleviation is
from about 7 to about 27 months.
Example 7
Treatment of Excessive Sweating
A male, age 65, with excessive unilateral sweating is treated by
administering 0.05 U/kg to about 2 U/kg of a modified neurotoxin,
depending upon degree of desired effect. Preferably the modified
neurotoxin comprises BoNT/E with an N-terminal myristylation site,
for example GVDIAY, fused to position 15 of its light chain, or a
position substantially corresponding to position 15 of the BoNT/A
light chain. The administration is to the gland nerve plexus,
ganglion, spinal cord or central nervous system. The specific site
of administration is to be determined by the physician's knowledge
of the anatomy and physiology of the target glands and secretary
cells. In addition, the appropriate spinal cord level or brain area
can be injected with the toxin. The cessation of excessive sweating
after the modified neurotoxin treatment is up to 27 months.
Example 8
Post Surgical Treatments
A female, age 22, presents a torn shoulder tendon and undergoes
orthopedic surgery to repair the tendon. After the surgery, the
patient is administered intramuscularly with about 0.05 U/kg to
about 2 U/kg of a modified neurotoxin to the shoulder. Preferably,
the modified neurotoxin comprises BoNT/A with an N-terminal
myristylation site, for example GLEVSF at position 254, deleted.
The specific site of administration is to be determined by the
physician's knowledge of the anatomy and physiology of the muscles.
The administered modified neurotoxin reduces movement of the arm to
facilitate the recovery from the surgery. The effect of the
modified neurotoxin is for about five weeks.
Example 9
Treatment of Spasmodic Dysphonia
A male, age 45, unable to speak clearly, due to spasm of the vocal
chords, is treated by injection of the vocal chords with a bout 0.1
U/kg to about 2 U/kg of modified neurotoxins according to the
present invention. After 3-7 days, the patient is able to speak
clearly. The patient's condition is alleviated for about 7 months
to about 27 months.
Example 10
Treatment of Spasmodic Torticollis
A male, age 45, suffering from spasmodic torticollis, as manifested
by spasmodic or tonic contractions of the neck musculature,
producing stereotyped abnormal deviations of the head, the chin
being rotated to the side, and the shoulder being elevated toward
the side at which the head is rotated, is treated by injection with
about 8 U/kg to about 15 U/kg of neurotoxins according to the
present invention. After 3-7 days, the symptoms are substantially
alleviated; i.e., the patient is able to hold his head and shoulder
in a normal position. The alleviation persists for about 7 months
to about 27 months.
Example 11
Treatment of Essential Tremor
A male, age 45, suffering from essential tremor, which is
manifested as a rhythmical oscillation of head or hand muscles and
is provoked by maintenance of posture or movement, is treated by
injection with about 8 U/kg to about 15 U/kg of modified neurotoxin
of the present invention. After two to eight weeks, the symptoms
are substantially alleviated; i.e., the patient's head or hand
ceases to oscillate. The symptoms are alleviated for about 5 months
to about 27 months.
Example 12
Production of a Modified Neurotoxin with an Altered Biological
Persistence
A modified neurotoxin according to the present invention may be
produced with recombinant techniques. An example of a recombinant
technique is one which includes the step of obtaining genetic
materials from oligonucleotide sequences having codes for a
modified neurotoxin according to the present invention. The genetic
constructs are incorporated into host cells for amplification by
first fusing the genetic constructs with a cloning vectors, such as
phages or plasmids. Then the cloning vectors are inserted into
hosts, preferably E. coli's. Following the expressions of the
recombinant genes in host cells, the resultant proteins can be
isolated using conventional techniques. See also International
Patent Application WO95/32738, the disclosure of which is
incorporated in its entirety by reference herein.
The modified neurotoxin produced according to this example has an
altered biological persistence. Preferably, the biological
persistence is enhanced, more preferably enhanced by about 20% to
about 300% relative to an identical neurotoxin without a
leucine-based motif.
Example 13
Modified Botulinum Toxins Having Additional Tyrosine
Phosphorylation Sites
In some embodiments, modified botulinum toxins of this invention
comprise one or more tyrosine phosphorylation sites in addition to
any naturally existing ones. In some embodiments, the modified
botulinum toxins comprise a heavy chain (modified or unmodified)
and a modified botulinum toxin light chain, wherein the modified
light chain comprises one or more tyrosine phosphorylation sites in
addition to any naturally existing ones. For example, the amino
acid sequence of a modified toxin, or the light chain thereof, of
the present invention is identical to that of a naturally existing
toxin, except for the added tyrosine phosphorylation site(s).
Without wishing to limit the invention to any theory or mechanism
of operation, it is believed that the additional tyrosine
phosphorylation site increases the biological persistence of the
modified botulinum toxin. Any tyrosine phosphorylation site may be
employed in accordance with the present invention. Non-limiting
examples of tyrosine phosphorylation sites include KLFERIY,
KLKFDKLY, and the tyrosine-based motif.
In some embodiments, a tyrosine-based motif may comprise four amino
acids. The amino acid at the N-terminal end of the tyrosine-based
motif can be a tyrosine. The amino acid at the C-terminal end of
the tyrosine-based motif can be a hydrophobic amino acid. The two
amino acids between the N-terminus and the C-terminus may be any
amino acid. In some embodiments, the tyrosine-based motif comprises
the sequence YKLL.
In some embodiments, a modified botulinum toxin of the present
invention comprises at least one tyrosine phosphorylation site,
e.g., a tyrosine-based motif, at the N-terminal of the light chain.
In some embodiments, a modified botulinum toxin of the present
invention comprises at least one tyrosine phosphorylation site,
e.g., a tyrosine-based motif, at the C-terminal of the light chain.
In some embodiments, a modified botulinum toxin of the present
invention comprises a tyrosine phosphorylation site, e.g.,
tyrosine-based motif, at the N-terminal and C-terminal of the light
chain.
Table 3 below shows the N-terminal and C-terminal regions of the
light chain of botulinum toxin types A-G. A modified botulinum
toxin of the present invention may comprise a tyrosine
phosphorylation site, e.g., a tyrosine-based motif, added to or
substituted into the N-terminal and/or C-terminal region of the
light chains of the respective botulinum toxins. In some
embodiments, a tyrosine phosphorylation site may substitute 1-10
consecutive amino acids of the light chain. In some embodiments,
the tyrosine phosphorylation site may substitute about 1-8, or
about 1-4, consecutive amino acids of the light chain.
TABLE-US-00003 TABLE 3 Toxin N-term (AAs 1-29) of LC C-term (last
50 AAs) of LC SEQ ID NO: BoNT/A PFVNKQFNYKDPVNGVDIAYIKI
GFNLRNTNLAANFNGQNTEINNM 47/48 PNAGQM NFTKLKNFTGLFEFYKLLCVRGI ITSK
BoNT/B PVTINNFNYNDPIDNDNIIMMEP YTIEEGFNISDKNMGKEYRGQNK 49/50 PFARGT
AINKQAYEEISKEHLAVYKIQMC KSVK BoNT/C1 PITINNFNYSDPVDNKNILYLDT
NIPKSNLNVLFMGQNLSRNPALR 51/52 HLNTLA KVNPENMLYLFTKFCHKAIDGRS LYNK
BoNT/D TWPVKDFNYSDPVNDNDILYLRI YTIRDGFNLTNKGFNIENSGQNI 53/54 PQNKLI
ERNPALQKLSSESVVDLFTKVCL RLTK BoNT/E PKINSFNYNDPVNDRTILYIKPG
GYNINNLKVNFRGQNANLNPRII 55/56 GCQEFY TPITGRGLVKKIIRFCKNIVSVK GIRK
BoNT/F PVAINSFNYNDPVNDDTILYMQI TVSEGFNIGNLAVNNRGQSIKLN 57/58 PYEEKS
PKIIDSIPDKGLVEKIVKFCKSV IPRK BoNT/G PVNIKXFNYNDPINNDDIIMMEP
QNEGFNIASKNLKTEFNGQNKAV 59/60 FNDPGP NKEAYEEISLEHLVIYRIAMCKP
VMYK
In some embodiments, the modified botulinum toxin comprises a
tyrosine phosphorylation site, e.g., a tyrosine-based motif, that
is located at the far most N-terminal region of the light chain of
the toxin. For example, a modified botulinum toxin type A may
comprise a light chain with an N-terminus starting as YKLLPFVNKQFNY
. . . (wherein YKLL is the tyrosine-based motif). In some
embodiments, the modified botulinum toxin comprises a tyrosine
phosphorylation site, e.g., tyrosine-based motif, that is located
within the first 1 to 10 amino acid residues of the N-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the first 5 to 15 amino acid residues of the N-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the first 10 to 20 amino acid residues of the N-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the first 15 to 29 amino acid residues of the N-terminal
region of the light chain.
In some embodiments, the modified botulinum toxin comprises a
tyrosine phosphorylation site, e.g., a tyrosine-based motif, that
is located at the far most C-terminal region of the light chain of
the toxin. For example, a modified botulinum toxin type A may
comprise a light chain with a C-terminus starting with . . .
YKLLCVRGIITSKYKLL (wherein YKLL is the tyrosine-based motif). In
some embodiments, the modified botulinum toxin comprises a tyrosine
phosphorylation site, e.g., tyrosine-based motif, that is located
within the last 1 to 10 amino acid residues of the C-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the last 5 to 15 amino acid residues of the C-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the last 10 to 20 amino acid residues of the C-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the last 15 to 25 amino acid residues of the C-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the last 20 to 30 amino acid residues of the C-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the last 25 to 35 amino acid residues of the C-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the last 30 to 40 amino acid residues of the C-terminal
region of the light chain. In some embodiments, the modified
botulinum toxin comprises a tyrosine-based motif that is located
within the last 35 to 50 amino acid residues of the C-terminal
region of the light chain.
In some embodiments, the modified botulinum toxin comprises a
modified light chain and a heavy chain of the same type of
botulinum toxin. In some embodiments, the modified botulinum toxin
is a chimera comprising a modified light chain of one botulinum
toxin type, and a heavy chain of another botulinum toxin type. The
heavy chain may further be modified.
Although the present invention has been described in detail with
regard to certain preferred methods, other embodiments, versions,
and modifications within the scope of the present invention are
possible. For example, a wide variety of modified neurotoxins can
be effectively used in the methods of the present invention in
place of clostridial neurotoxins. Also, the corresponding genetic
codes, i.e. DNA sequence, to the modified neurotoxins are also
considered to be part of this invention. Additionally, the present
invention includes peripheral administration methods wherein two or
more modified neurotoxins, for example BoNT/E fused with a
modification site and BoNT/B fused with a modification site, are
administered concurrently or consecutively. Furthermore, a
"targeting component" may be added to or substituted onto a
modified neurotoxin of this invention. The "targeting component"
may be a small molecule or a polypeptide having selective binding
to a particular receptor. As such, a modified neurotoxin of the
present invention comprising a targeting component may be
specifically directed to a specific target receptor. See Foster et
al in U.S. Pat. No. 5,989,545 and Donovan in U.S. patent
application Ser. No. 09/489,667, the disclosures of which are
incorporated herein by reference.
While this invention has been described with respect to various
specific examples and embodiments, it is to be understood that the
invention is not limited thereto and that it can be variously
practiced with the scope of the following claims.
SEQUENCE LISTINGS
1
6014PRTClostridium botulinumSITE(1)...(4)N-glycosylation site 1Asn
Leu Thr Arg 124PRTClostridium botulinumSITE(1)...(4)N-glycosylation
site 2Asn Tyr Thr Ile 134PRTClostridium
botulinumSITE(1)...(4)N-glycosylation site 3Asn Phe Thr Lys
144PRTClostridium botulinumSITE(1)...(4)N-glycosylation site 4Asn
Phe Thr Gly 154PRTClostridium botulinumSITE(1)...(4)Casein kinase
II phosphorylation site 5Thr Asn Pro Glu 164PRTClostridium
botulinumSITE(1)...(4)Casein kinase II phosphorylation site 6Ser
Tyr Tyr Asp 174PRTClostridium botulinumSITE(1)...(4)Casein kinase
II phosphorylation site 7Thr Asp Asn Glu 184PRTClostridium
botulinumSITE(1)...(4)Casein kinase II phosphorylation site 8Ser
Thr Ile Asp 194PRTClostridium botulinumSITE(1)...(4)Casein kinase
II phosphorylation site 9Ser Gly Leu Glu 1104PRTClostridium
botulinumSITE(1)...(4)Casein kinase II phosphorylation site 10Ser
Phe Glu Glu 1114PRTClostridium botulinumSITE(1)...(4)Casein kinase
II phosphorylation site 11Ser Leu Gln Glu 1124PRTClostridium
botulinumSITE(1)...(4)Casein kinase II phosphorylation site 12Thr
Ile Tyr Asp 1136PRTClostridium botulinumSITE(1)...(6)N-terminal
myristylation site 13Gly Val Asp Ile Ala Tyr 1 5146PRTClostridium
botulinumSITE(1)...(6)N-terminal myristylation site 14Gly Ser Tyr
Arg Ser Glu 1 5156PRTClostridium botulinumSITE(1)...(6)N-terminal
myristylation site 15Gly Leu Glu Val Ser Phe 1 5163PRTClostridium
botulinumSITE(1)...(3)Protein kinase C phosphorylation site 16Ser
Tyr Arg 1173PRTClostridium botulinumSITE(1)...(3)Protein kinase C
phosphorylation site 17Ser Gly Lys 1183PRTClostridium
botulinumSITE(1)...(3)Protein kinase C phosphorylation site 18Thr
Ser Lys 1197PRTClostridium botulinumSITE(1)...(7)Tyrosine
phosphorylation site 19Lys Leu Phe Glu Arg Ile Tyr 1
5208PRTClostridium botulinumSITE(1)...(8)Tyrosine phosphorylation
site 20Lys Leu Lys Phe Asp Lys Leu Tyr 1 5214PRTClostridium
botulinumSITE(1)...(4)N-glycosylation site 21Asn Leu Ser Gly
1224PRTClostridium botulinumSITE(1)...(4)N-glycosylation site 22Asn
Gly Ser Gly 1234PRTClostridium
botulinumSITE(1)...(4)N-glycosylation site 23Asn Ser Ser Asn
1244PRTClostridium botulinumSITE(1)...(4)N-glycosylation site 24Asn
Ile Ser Leu 1254PRTClostridium
botulinumSITE(1)...(4)N-glycosylation site 25Asn Asp Ser Ile
1264PRTClostridium botulinumSITE(1)...(4)N-glycosylation site 26Asn
Ile Ser Glu 1274PRTClostridium botulinumSITE(1)...(4)Casein kinase
II phosphorylation site 27Thr Pro Gln Asp 1284PRTClostridium
botulinumSITE(1)...(4)Casein kinase II phosphorylation site 28Ser
Tyr Tyr Asp 1294PRTClostridium botulinumSITE(1)...(4)Casein kinase
II phosphorylation site 29Ser Asp Glu Glu 1304PRTClostridium
botulinumSITE(1)...(4)Casein kinase II phosphorylation site 30Ser
Ala Val Glu 1314PRTClostridium botulinumSITE(1)...(4)Casein kinase
II phosphorylation site 31Ser Met Asn Glu 1324PRTClostridium
botulinumSITE(1)...(4)Casein kinase II phosphorylation site 32Thr
Asn Ile Glu 1334PRTClostridium botulinumSITE(1)...(4)Casein kinase
II phosphorylation site 33Ser Phe Thr Glu 1344PRTClostridium
botulinumSITE(1)...(4)Casein kinase II phosphorylation site 34Thr
Glu Phe Asp 1356PRTClostridium botulinumSITE(1)...(6)N-terminal
myristylation site 35Gly Leu Tyr Gly Ala Lys 1 5366PRTClostridium
botulinumSITE(1)...(6)N-terminal myristylation site 36Gly Thr Asp
Leu Asn Ile 1 5376PRTClostridium botulinumSITE(1)...(6)N-terminal
myristylation site 37Gly Gln Asn Ala Asn Leu 1 5383PRTClostridium
botulinumSITE(1)...(3)Protein kinase C phosphorylation site 38Ser
Leu Lys 1393PRTClostridium botulinumSITE(1)...(3)Protein kinase C
phosphorylation site 39Ser Leu Arg 1403PRTClostridium
botulinumSITE(1)...(3)Protein kinase C phosphorylation site 40Ser
Phe Arg 1413PRTClostridium botulinumSITE(1)...(3)Protein kinase C
phosphorylation site 41Thr Thr Lys 1423PRTClostridium
botulinumSITE(1)...(3)Protein kinase C phosphorylation site 42Thr
Gln Lys 1433PRTClostridium botulinumSITE(1)...(3)Protein kinase C
phosphorylation site 43Thr Gly Arg 1443PRTClostridium
botulinumSITE(1)...(3)Protein kinase C phosphorylation site 44Ser
Val Lys 1457PRTClostridium botulinumSITE(1)...(7)Tyrosine kinase
phosphorylation site 45Lys Asn Gly Asp Ser Ser Tyr 1
5468PRTClostridium botulinumSITE(1)...(8)Tyrosine kinase
phosphorylation site 46Lys Asp Val Phe Glu Ala Lys Tyr 1
54729PRTClostridium botulinumDOMAIN(1)...(29)N-terminal amino acids
of BoNT/A light chain 47Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp
Pro Val Asn Gly Val 1 5 10 15Asp Ile Ala Tyr Ile Lys Ile Pro Asn
Ala Gly Gln Met 20 254850PRTClostridium
botulinumDOMAIN(1)...(50)C-terminal amino acids of BoNT/A light
chain 48Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn Phe Asn Gly
Gln 1 5 10 15Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu Lys
Asn Phe Thr 20 25 30Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg
Gly Ile Ile Thr 35 40 45Ser Lys 504929PRTClostridium
botulinumDOMAIN(1)...(29)N-terminal amino acids of BoNT/B light
chain 49Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn
Asp 1 5 10 15Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr 20
255050PRTClostridium botulinumDOMAIN(1)...(50)C-terminal amino
acids of BoNT/B light chain 50Tyr Thr Ile Glu Glu Gly Phe Asn Ile
Ser Asp Lys Asn Met Gly Lys 1 5 10 15Glu Tyr Arg Gly Gln Asn Lys
Ala Ile Asn Lys Gln Ala Tyr Glu Glu 20 25 30Ile Ser Lys Glu His Leu
Ala Val Tyr Lys Ile Gln Met Cys Lys Ser 35 40 45Val Lys
505129PRTClostridium botulinumDOMAIN(1)...(29)N-terminal amino
acids of BoNT/C1 light chain 51Pro Ile Thr Ile Asn Asn Phe Asn Tyr
Ser Asp Pro Val Asp Asn Lys 1 5 10 15Asn Ile Leu Tyr Leu Asp Thr
His Leu Asn Thr Leu Ala 20 255250PRTClostridium
botulinumDOMAIN(1)...(50)C-terminal amino acids of BoNT/C1 light
chain 52Asn Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn
Leu 1 5 10 15Ser Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn
Met Leu Tyr 20 25 30Leu Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly
Arg Ser Leu Tyr 35 40 45Asn Lys 505329PRTClostridium
botulinumDOMAIN(1)...(29)N-terminal amino acids of BoNT/D light
chain 53Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp
Asn 1 5 10 15Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile 20
255450PRTClostridium botulinumDOMAIN(1)...(50)C-terminal amino
acids of BoNT/D light chain 54Tyr Thr Ile Arg Asp Gly Phe Asn Leu
Thr Asn Lys Gly Phe Asn Ile 1 5 10 15Glu Asn Ser Gly Gln Asn Ile
Glu Arg Asn Pro Ala Leu Gln Lys Leu 20 25 30Ser Ser Glu Ser Val Val
Asp Leu Phe Thr Lys Val Cys Leu Arg Leu 35 40 45Thr Lys
505529PRTClostridium botulinumDOMAIN(1)...(29)N-terminal amino
acids of BoNT/E light chain 55Pro Lys Ile Asn Ser Phe Asn Tyr Asn
Asp Pro Val Asn Asp Arg Thr 1 5 10 15Ile Leu Tyr Ile Lys Pro Gly
Gly Cys Gln Glu Phe Tyr 20 255650PRTClostridium
botulinumDOMAIN(1)...(50)C-terminal amino acids of BoNT/E light
chain 56Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe Arg Gly Gln Asn
Ala 1 5 10 15Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr Gly Arg
Gly Leu Val 20 25 30Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val Ser
Val Lys Gly Ile 35 40 45Arg Lys 505729PRTClostridium
botulinumDOMAIN(1)...(29)N-terminal amino acids of BoNT/F light
chain 57Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp
Asp 1 5 10 15Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser 20
255850PRTClostridium botulinumDOMAIN(1)...(50)C-terminal amino
acids of BoNT/F light chain 58Thr Val Ser Glu Gly Phe Asn Ile Gly
Asn Leu Ala Val Asn Asn Arg 1 5 10 15Gly Gln Ser Ile Lys Leu Asn
Pro Lys Ile Ile Asp Ser Ile Pro Asp 20 25 30Lys Gly Leu Val Glu Lys
Ile Val Lys Phe Cys Lys Ser Val Ile Pro 35 40 45Arg Lys
505929PRTClostridium botulinumDOMAIN(1)...(29)N-terminal amino
acids of BoNT/G light chain 59Pro Val Asn Ile Lys Xaa Phe Asn Tyr
Asn Asp Pro Ile Asn Asn Asp 1 5 10 15Asp Ile Ile Met Met Glu Pro
Phe Asn Asp Pro Gly Pro 20 256050PRTClostridium
botulinumDOMAIN(1)...(50)C-terminal amino acids of BoNT/G light
chain 60Gln Asn Glu Gly Phe Asn Ile Ala Ser Lys Asn Leu Lys Thr Glu
Phe 1 5 10 15Asn Gly Gln Asn Lys Ala Val Asn Lys Glu Ala Tyr Glu
Glu Ile Ser 20 25 30Leu Glu His Leu Val Ile Tyr Arg Ile Ala Met Cys
Lys Pro Val Met 35 40 45Tyr Lys 50
* * * * *